Leishmaniasis is a vector-borne zoonotic disease endemic to the Middle East, Asia, Africa and Central America. C3HeB/FeJ (C3H) mice infected with Leishmania major develop a CD4+ TH1 response and resolve infection, while infection with Leishmania amazonensis stimulates a poor T cell response, resulting in chronic disease. When C3H mice are co-infected with both species of Leishmania we observe a healing phenotype similar to infection with L. major alone. In contrast, co-infected C57BL/6 (B6) mice have a non-healing phenotype similar to infection with L. amazonensis. We have developed an in vitro assay and have found that CD4+ T cells and B cells from the draining lymph node L. major-infected C3H mice placed in this transwell assay are sufficient and necessary for macrophage activation and killing of L. amazonensis. When B6 mouse-derived total lymph node cells or purified CD4+ T cells and B cells from a B6 mouse infected with L. major are co-cultured in vitro with L. amazonensis infected macrophages parasite killing is not observed. A series of cell transfer studies have demonstrated that B cells from the B6 mice do not promote killing of intracellular L. amazonensis. These findings from our in vitro parasite killing assay and in vivo transfer experiments indicate that B cells play a prominent role in the cell mediated immune response against L. amazonensis and that B cells from B6 mice are unable to promote macrophage parasite killing compared to cells from CSH mice. We have generated two hypotheses concerning the difference between C3H and B6 B cells. First, we hypothesize B cells from C3H mice produce pro-inflammatory cytokines that positively influence CD4+ T cells to activate co-infected macrophages, while B cells from B6 mice produce anti-inflammatory cytokines that negatively influence CD4+ T cells to activate infected macrophages. Our second hypothesis is the production of the novel antibody isotype lgG2c by B6 mice may inadequately stimulate infected macrophages via the Fc gamma receptors. The work in this proposal will 1) establish if B cells from C57BL/6 mice inadequately stimulate a productive Th1 immune response during co-infection by phenotypically characterizing B cells;and 2) determine if lgG2c production by B6 B cells alters macrophage activation. Results of this work will emphasize the importance of B cells in Leishmania infection and all will provide a foundation for development of treatment and vaccine options of cutaneous leishmaniasis in animals and humans. These findings will also advance our understanding of genetic predispositions to leishmaniasis.